Aneuploidy, an incorrect chromosome number, is a common trait of cancer. However, the genetic defects and molecular mechanisms underlying chromosome instability, and the role of aneuploidy in the etiology of cancer remain poorly understood. Bub1, a mitotic checkpoint gene encoding a multi-functional serine/threonine protein kinase, has emerged as a prominent cancer-associated gene, with both up- and downregulation causing tumors in mice. Although aberrant expression in both directions has been observed in human tumors, the discovery that high Bub1 expression is a biomarker for poor clinical outcome for various tumor types implies that this alteration may be particularly relevant in tumorigenesis. The goal of this application is to determine how mechanistically Bub1 overexpression drives neoplastic transformation, and the extent to which it is causally linked to tumor aggressiveness and metastasis, with the long-term goal to exploit Bub1 or its downstream effectors for preventive and therapeutic purposes. As a first step toward these goals, we engineered two novel Bub1 mouse models, one overexpressing Bub1 and one lacking Bub1 kinase activity. Initial characterization of these models suggests a novel function of Bub1 as a master regulator of Aurora B, a key component of the attachment error correction machinery that prevents chromosome missegregation by resolving aberrant microtubule-kinetochore attachments. Our central hypothesis is that increased Bub1 kinase activity drives tumor development and aggressiveness through hyperactivation of Aurora B kinase. Guided by persuasive preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine how mechanistically Bub1 activates Aurora B and the extent to which this mechanism is implicated in tumor development; and 2) Determine the extent to which Bub1 overexpression drives tumor aggressiveness and cooperates with other CIN gene defects. In the first aim, we will use already established and newly designed Bub1 mutant mouse models in combination with small molecule inhibitors of Aurora B to resolve the molecular mechanism by which Bub1 regulates Aurora B, and test whether deregulation of this mechanism is critical for Bub1's oncogenic properties. In the second aim, we will use Bub1 transgenic mice in combination with the Errb2 breast cancer model to test whether Bub1 overexpression is not just a biomarker for poor prognosis but in fact drives tumor invasiveness and metastasis. In addition, we will explore the novel concept that Bub1 overexpression synergizes with other CIN genes that predict poor clinical outcome. The expected overall impact of this project is that it will fundamentally advance our mechanistic understanding of the normal and neoplastic functions of a prominent cancer-causing CIN gene, and provide proof for the novel concepts that CIN gene alterations that predict poor prognosis can drive tumor aggressiveness and synergize with one another. Knowledge from these efforts could be exploited to devise novel strategies for cancer treatment and prevention in addition to conceptually advancing the fields of mitosis and cancer biology. PUBLIC HEALTH RELEVANCE: Cancer cells are characterized by an abnormal number of chromosomes, but the genetic and molecular defects driving this condition and its precise role in the development of cancer remain poorly understood, which is a critical barrier to the development of novel cancer intervention strategies. Our goal is to fill this gap in knowledge through a series of innovative studies of Bub1, a prominent chromosomal instability gene that is overexpressed in various human cancer types and associated with poor clinical prognosis.